Microorganisms are today widely used for protein or DNA production. Usually, these processes require the use of bacterial plasmids as vectors carrying the gene to be expressed. However, plasmid maintenance within microorganisms is limited, and plasmid instability thus represents a significant concern in recombinant protein production or in DNA production.
It has been demonstrated that the growth rate of plasmid-bearing cells is significantly reduced relative to that of a plasmid-free cell. One theory is that plasmid replication and transcription, as well as protein production, represent a significant burden on cellular metabolism. Hence, in a fermentation process, cells losing the plasmid exhibit a higher fitness than cells still bearing the plasmid, and the former rapidly overcome the latter in the bacterial population.
In order to limit plasmid loss in a cell population, and to avoid plasmid-free cells to survive and dominate the culture, selectable markers were inserted in plasmids.
The most common selectable markers used in fermentation procedures are antibiotic-resistance genes. However, contamination of the product or biomass by antibiotics (or genes encoding an antibiotic resistance) is unacceptable from a medical or regulatory perspective. Moreover, antibiotic-resistance genes may propagate in the environment, or be transferred to pathogenic strains. Moreover, recent studies demonstrated that using antibiotic-resistance genes for producing a recombinant protein strongly reduces the yield of protein production (Peubez et al., Microbial Cell factories, 2010, 9:65).
There is thus a need for other systems allowing plasmid maintenance, and free from drawbacks of antibiotic-resistance genes.
An alternative to the use of antibiotic-resistance genes is the complementation of an essential mutated chromosomal gene by a wild-type allele inserted into the plasmid. For example, systems were developed in which the mutant host is unable to synthesize an essential amino acid without a plasmid carrying out the gene that provides this function. However, this approach seriously restricts the possible choices in growth medium.
Another strategy developed is a system in which a plasmid-mediated repressor titration overcomes the repression of an essential chromosomal gene placed under the control of the lac operator. However, this procedure has the following limitations: (i) it makes the Lac promoter unavailable for other purposes such as protein expression, (ii) the system is limited to E. coli or other bacteria wherein the Lac promoter is functional, and (iii) medium containing lactose has to be avoided.
Another alternative system to the use of antibiotic-resistance genes is based on couples of poison proteins (i.e. molecules which are toxic for the host cell) combined to their antidotes. For example, the poison gene may be expressed by the host cell from a chromosomal copy, while the antidote is carried by the plasmid. Therefore, presence of the plasmid is required for the host cell survival. An example of couple poison/antidote is the ccdA (antidote)/ccdB (poison) system. CcdA and CcdB are the antidote and toxin proteins encoded by the E. coli F plasmid. Together, they ensure the death of daughter cells that do not receive a copy of F. Expression of the ccdB protein interferes with the rejoining step of DNA gyrase, causing the host cell chromosome to be cut to pieces. Other examples of antidote/poison couples include, but are not limited to, Kis/Kid proteins, Phd/Doc proteins, RelB/relE proteins, PasB (or PasC)/PasA proteins, mazF/maze proteins.
This antidote/poison system has been extensively used in cloning methods, as described for example in the U.S. Pat. No. 8,470,580: a host cell comprising the gene encoding a poison is used in combination with a plasmid comprising the gene encoding the corresponding antidote. This system thus allows the direct selection of cells having integrated a gene of interest, as only cells expressing the antidote gene survive.
This system is also used for protein production, as previously described by the inventors (Szpirer and Milinkovitch, BioTechniques, 2005, 38(5):775-781). Unexpectedly, an increase of three to five-fold of the recombinant protein production level was observed, demonstrating the great potential of this system for producing recombinant proteins.
However, the Inventors observed that, when this system is used for producing toxic proteins, some drawbacks may appear, such as, for example, mutations within the gene of interest, mutations in the antidote/poison system and the like. Willing to develop an improved plasmid stabilization system, they modified the classic system comprising one copy of the antidote gene combined with one copy of the poison gene. Surprisingly and unexpectedly, they showed that the insertion of an additional copy of the poison gene in the genome of the host cell increases the stability of the plasmid, but also increases the yield of protein production (in a non-correlate manner).